The Gotham Rail is an elevated rail system threaded through the various districts of Gotham City.
- Due to the continuing chaos and destruction plaguing the city, the rail system has been torn apart; with sections of rail line missing and or destroyed thanks to Brainiac's incursion.
- As the Penguin's thugs attack the player from a bridge in the Gotham Old Subway, a Gotham Rail train arrives and knocks a group of thugs off the bridge.
- In Gotham Under Siege, the Amazon forces use the decommissioned rail line to cross the Sprang River. Wonder Woman's rebel forces use the western-most bridge to cross south while Hippolyta's loyalists use the central bridge to cross north.
- The Gotham Rail is based in part on the Chicago "L" rapid transit system.